Inhibition of mucin release from airway goblet cells by polycationic peptides

ABSTRACT

Polycationic peptides have been shown to be effective inhibitors of mucin secretion. Inhibition of mucin secretion using these polycationic peptides may be an important tool in the treatment of diseases associated with mucin hypersecretion, including asthma, chronic bronchitis, cystic fibrosis, and bronchiectasis.

BACKGROUND OF THE INVENTION

[0001] Hypersecretion of mucin in the airways is associated with avariety of diseases, including asthma, chronic bronchitis, cysticfibrosis, and bronchiectasis. Effective measures for inhibiting mucinsecretion in the airways would be useful to mitigate the deleteriouseffects associated with mucin hypersecretion. Effective inhibition ofmucin secretion would also be useful in enhancing the delivery oftherapeutic agents to the airways and via the airways.

SUMMARY OF THE INVENTION

[0002] It has been discovered, according to this invention, thatpolycationic peptides inhibit mucin secretion from airway goblet cells.It has also been discovered that the inhibition of mucin secretion bypolycationic peptides is accompanied by minimal cytotoxicity to cells ofairways, thereby by indicating the suitability of polycationic peptidesfor use in animals, particularly humans, for the inhibition of mucinsecretion. It has also been discovered that polycationic peptides canprevent S02 induced goblet cell metaplasia.

[0003] It is contemplated as part of this invention that polycationicmolecules, particularly polycationic peptides or peptide mimetics, maybe employed to inhibit mucin secretion for a variety of therapeuticpurposes. For example, the polycationic molecules may be administered toalleviate mucin hypersecretion, particularly in disease conditionsassociated with mucin hypersecretion. For example, the compositions ofthe invention may be administered to treat asthma, chronic bronchitis,cystic fibrosis, bronchiectasis, and chronic obstructive pulmonarydisease. As another example, the polycationic molecules may beadministered to reduce mucin in the airways in order to facilitate thebioavailability of therapeutic agents targeted to the airways, such as,for example, bronchodialators. As another example, the polycationicmolecules may be administered to reduce mucin in the airways, therebyminimizing airway impedance and facilitating therapeutic agent deliveryto the alveoli or through the alveoli to the blood stream.

[0004] Treat, treated or treatment, as used herein, means theadministration of polycationic molecules to the airways of an animal toprevent, mitigate, alleviate or cure a disease or the symptoms of adisease.

[0005] Animal, as used herein, means any vertebrate animal, includinghumans, farm animals and pets.

[0006] Airways or airway, as used herein, refers to any part of thelungs that is capable of mucin production.

[0007] Polycationic molecules that may be used in accordance with thisinvention include, but are not limited to, polycationic peptides andpolycationic peptide mimetics. Polycationic peptides are a preferredpolycationic molecule because they can be easily degraded to aminoacids.

[0008] The polycationic peptides typically comprise about 5 to about 60amino acids, preferably about 5 to about 40 amino acids, and morepreferably about 10 to about 25 amino acids. Smaller molecules arepreferred because of greater ease of handling. A polycationic peptidetypically possesses a sufficient number of positively charged aminoacids such that the pKa of the peptide is greater than 9.0, preferablygreater than 10.0, and more preferably greater than 11.0. Typically, atleast about 20% of the amino acid residues are positively charged,preferably at least about 40%, more preferably at least about 60%, andmost preferably at least about 80%. Positively charged amino acidsinclude, for example, lysine, arginine, or ornithine. Positively chargedrefers to the side chains of the amino acids which have a net positivecharge at a pH 7.0. The effectiveness of any particular polycationicmolecule in inhibiting mucin, with minimal toxicity, may be established,for example, using techniques and models described herein. At an optimalconcentration, a polycationic molecule, polycationic peptide, orpolycationic peptide mimetic, according to this invention, preferablyinhibits mucin secretion at least about 50%, more preferably at leastabout 70%, and most preferably at least about 90%. The degree ofinhibition of mucin secretion may be determined, for example, using ahamster tracheal surface epithelial (HTSE) cell culture system asdescribed in the Examples herein. The degree of inhibition of mucinsecretion may also be determined using other techniques, including invitro techniques such as are described, for example, in Adler et al.,1990, J. Clin. Invest. 85:75-85 and in Adler et al., 1992, Am. J.Respir. Cell Mol. Biol. 6:550-556 and including in vivo techniques suchas are described, for example, in Temann et al., 1997, Am. J. Respir.Cell Mol. Biol. 16:471-478 and Fahy et al., 1993, Am. Rev. Respir. Dis.147:1132-1137. A polycationic molecule, polycationic peptide, orpolycationic peptide mimetic according to this invention preferably hasminimal cytotoxicity. Cytotoxicity may be determined, for example, usingan LDH release assay, a ⁵¹Cr release assay, or a cell exfoliation assayas described in the examples. Cytotoxicity may also be determined, forexample, using an HTSE cell culture system as described in the examples.In determining cytotoxicity for polycationic molecules of the inventionusing the above listed assays, LDH release, ⁵¹Cr release, or cellexfoliation is typically less than about 115% of that observed incontrol cells, preferably less than about 110%, and more preferably lessthan about 105%.

[0009] Preferred polycationic peptides for use in accordance with thisinvention are poly-L-lysine, poly-L-arginine, or poly-L-lysine andpoly-L-arginine heteropolymers. Polycationic peptides containing otherpositively charged amino acid residues such as, for example,poly-L-ornithine, may also be employed. The amino acids of thepolycationic peptides may be naturally occurring proteogenic amino acidsas well as non-naturally occurring amino acids such as amino acidanalogs. One of skill in the art would know that this definitionincludes, unless otherwise indicated, naturally occurring proteogenic(D) or (L) amino acids, chemically modified amino acids, including aminoacid analogs such as penicillamine (3-mercapto-D-valine), naturallyoccurring non-proteogenic amino acids such as norleucine and chemicallysynthesized compounds that have properties known in the art to becharacteristic of an amino acid. As used herein, the term “proteogenic”indicates that the amino acid can be incorporated into a protein in acell through well-known metabolic pathways. The choice of including an(L)- or a (D)-amino acid into a peptide of the present inventiondepends, in part, on the desired characteristics of the peptide. Forexample, the incorporation of one or more (D)-amino acids can conferincreasing stability on the peptide in vitro or in vivo. As used herein,the term “amino acid equivalent” refers to compounds which depart fromthe structure of the naturally occurring amino acids, but which havesubstantially the structure of an amino acid, such that they can besubstituted within a peptide which retains biological activity. Thus,for example, amino acid equivalents can include amino acids having sidechain modifications or substitutions, and also include related organicacids, amides or the like. The term “amino acid” is intended to includeamino acid equivalents. The term “residues” refers both to amino acidsand amino acid equivalents.

[0010] As used herein, the term “peptide” is used in its broadest senseto refer to compounds containing amino acid equivalents or othernon-amino groups, while still retaining the desired functional activityof inhibiting mucin secretion. Peptide equivalents can differ fromconventional peptides by, for example, the replacement of one or moreamino acids with related organic acids (such as PABA) or thesubstitution or modification of side chains or functional groups. It isto be understood that limited modifications can be made to a peptidewithout destroying its biological function, such as for example, theaddition of chemical moieties such as amino or acetyl groups.

[0011] Polycationic peptides that may be used in accordance with thisinvention include naturally occurring peptides, synthetic peptides, andanalogs thereof. The effectiveness of any particular polycationicpeptide in inhibiting mucin secretion can be established, for example,by using the techniques described herein.

[0012] Polycationic peptides useful for this invention may be producedusing techniques that are well known in the art, including chemicalsynthesis techniques and recombinant DNA techniques. The production ofpolycationic peptides using recombinant DNA techniques is described, forexample, in U.S. Pat. No. 5,593,866. Chemical synthesis of peptides canbe accomplished using techniques that are well known in the art, such asTBOC or FMOC protection of alpha-amino groups. (See, Coligan, et al.,Current Protocols in Immunology, Wiley Interscience, 1991, Unit 9).Peptides of the invention can also be synthesized by the well knownsolid phase peptide synthesis methods (Merrifield, J. Am. Chem. Soc.,85:2149 (1962); Stewart and Young, Solid Phase Peptide Synthesis(Freeman, San Francisco, 1969) pp. 27-62).

[0013] Non-peptide compounds that mimic the mucin inhibiting function ofpolycationic peptides, peptide mimetics, can also be employed inconjunction with the invention. Such peptide mimetics can be produced asdescribed, for example, by Saragovi et al., Science 253:792-95 (1991).Peptide mimetics are molecules which mimic elements of protein secondarystructure. See, for example, Johnson et al., Peptide Turn Mimetics, inBiotechnology and Pharmacy, Pezzuto et al., Eds., (Chapman and Hall, NewYork 1993). The underlying rationale behind the use of peptide mimeticsis that the peptide backbone of proteins exists chiefly to orient aminoacid side chains in such a way as to facilitate molecular interactions.For the purposes of the present invention, appropriate peptide mimeticsare considered to be the equivalent of mucin inhibiting polycationicpeptides.

[0014] If the compounds described above are employed, the skilledartisan can routinely ensure that such compounds are amenable for usewith the present invention in view of the mucin inhibition assays andcell toxicity assays described herein.

[0015] It is also contemplated as part of the invention that lowmolecular weight polyanions such as, for example, low molecular weightheparin may be employed to modulate the effect of the polycationicmolecules in inhibiting mucin release, or to mitigate cytotoxcity of thepolycationic peptides.

[0016] The invention also includes various pharmaceutical compositionsand pharmaceutical articles of manufacture that may be employed toinhibit mucin secretion in the airways of an animal. The pharmaceuticalcompositions according to the invention are prepared by bringing apolycationic molecule into a form suitable for administration to asubject using adjuvants, carriers, excipients and additives orauxiliaries, or devices. Pharmaceutically acceptable adjuvants,carriers, excipients, additives or auxiliaries are described, forexample, in Remington's Pharmaceutical Sciences, 15th ed. Easton: MackPublishing Co. (1990), the contents of which are hereby incorporated byreference. The pH and exact concentration of the various components ofthe pharmaceutical composition are adjusted according to routine skillsin the art. See Goodman and Gilman's The Pharmacological Basis forTherapeutics (7th ed.). Particularly preferred adjuvants for use inconjunction with the invention are inhalant adjuvants. An inhalantadjuvant, as used herein, refers to any composition which facilitatesthe airborne administration of the polycationic molecules to the airwaysof an animal.

[0017] The polycationic peptides may be administered to the airways inany number of ways that are well known in the art, including viainhalation or via physical application, including the use of abronchoscope. A preferred method for administration is by inhalation,including for example, both oral inhalation and nasal inhalation. Forinhalation, the compounds of the invention are conveniently deliveredfrom an insufflator, a nebulizer, a pump, a pressurized pack, or otherconvenient means of delivering an aerosol or non-aerosol spray of apowder or a liquid. Pressurized packs may comprise a suitable propellantsuch a liquefied gas or a compressed gas. Liquefied gases include, forexample, fluorinated chlorinated hydrocarbons, hydrochlorofluorocarbons,hydrochlorocarbons, hydrocarbons, and hydrocarbon ethers. Compressedgases include, for example, nitrogen, nitrous oxide, and carbon dioxide.In particular, the use of dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas is contemplated. In the case of a pressurizedaerosol, the dosage unit may be determined by providing a valve todeliver a metered amount. Alternatively, for administration byinhalation or insufflation, the compounds of the invention may be in theform of a dry powder composition, for example, a powder mix of thecompound and a suitable powder base such as lactose or starch. Thepowder composition may be presented in unit dosage form such as, forexample, capsules, cartridges, or blister packs from which the powdermay be administered with the aid of an inhalator or insufflator.

[0018] In accordance with the invention, the compounds of the inventionmay be administered to inhibit mucin secretion in conjunction with othertherapeutic agents. For example, for the treatment of asthma,polycationic polypeptides may be administered in conjunction withsympathomimetic bronchodilators, anticholinergic bronchodilators,theophylline bronchodialators, corticosteroids, and antimediators.

[0019] It will be appreciated that the amount of the polycationicmolecule or the polycationic peptide to be administered in conjunctionwith the invention will vary depending upon the condition and the animalbeing treated. Suitable dosage amounts can be routinely determined byone of ordinary skill in the art. The desired daily dose may bepresented in single dose or as divided doses administered at appropriateintervals, such as two, three, four or more sub-doses per day.

BRIEF DESCRIPTION OF THE FIGURES

[0020]FIG. 1 is a bar graph showing the effects of poly-L-arginine (PLA)poly-L-lysine (PLL) on mucin release in HTSE cells. Confluent cells in16 mm wells were metabolically radiolabeled with ³H-glucosamine for 24hr and chased for 30 min in the presence of varying concentrations ofeither PLA or PLL. The amount of ³H-mucins in the spent medium wasmeasured. Each bar represents a mean ±S.E.M from four culture wells. An(*) indicates a value which is significantly different (p<0.05) from acontrol value, based on the Student's t-test for unpaired samples.

[0021]FIG. 2 is a bar graph showing the effects of PLA and PLL on LDHrelease from HTSE cells. Confluent cells in 16 mm wells were treatedwith varying concentrations of either PLA or PLL for 30 min, andaliquots of the spent media were collected and assayed for LDH levels.Each bar represents a mean ±S.E.M from four culture wells. There was nosignificant difference (p>0.05) among different concentration groups,based on the Student's t-test for unpaired samples.

[0022]FIG. 3 is a bar graph showing the effects of low molecular weightheparin (LMWH) in inhibiting the inhibitory effect of polycationicpeptides on mucin release in HTSE cells. Confluent cells in 16 mm wellswere metabolically radiolabeled with ³H-glucosamine for 24 hrs andpretreated with 2×10⁻⁵ M LMWH for 5 min prior to chasing for 30 min inthe presence of either 10⁻⁵ M PLA or 10⁻⁵ M PLL. The amount of ³H-mucinsin the spent medium was measured. Each bar represents a mean±S.E.M. fromfour culture wells. A (P) represents polycationic peptides and an (H)represents low molecular weight heparin. An (*) indicates a value thatis significantly different (p<0.05) from a control value based on theStudent's t-test for unpaired samples.

[0023]FIG. 4 is a graph indicating the results of CM-sepharosecation-exchange chromatography of polycationic peptides followingN-acetylation. A solution of PLA at a concentration of 10⁻⁴ M wasN-acetylated using acetic anhydride, and the acetylated and the original(unacetylated) forms of PLA were separated by CM-Sepharosecation-exchange chromatography. The unacetylated PLA was retained on thecolumn (Panel A) and the N-acetylated PLA was eluted from the column(Panel B). The yield of N-acetylated PLA was greater than 95%.

[0024]FIG. 5 is a bar graph showing the effects of N-acetylated PLA andN-acetylated PLL on mucin release by HTSE cells. Confluent cells in 16mm wells were metabolically radiolabeled with ³H-glucosamine for 24 hrand chased for 30 min in the presence of 10⁻⁵ M of polycationicpeptides. The amount of ³H-mucins in the spent medium was measured. P:original formulations of the unacetylated polycationic peptides, withoutcolumn purification. N-AcP: N-acetylated polycationic peptides (columnpurified). P-P: unacetylated polycationic peptides that were columnpurified. Each bar represents a mean ±S.E.M. from four culture wells. Anasterisk (*) indicates a value that is significantly different (p<0.05)from the control value based on the Student's t-test for unpairedsamples.

[0025]FIG. 6 is a bar graph showing the effect of polycationic peptideson ATP-induced mucin release in HTSE cells. Confluent cells in 16 mmwells were metabolically radiolabeled with ³H-glucosamine for 24 hr andchased for 30 min in the presence of a combination of ATP, at aconcentration of 2×10⁻⁴ M, and polycationic peptides, at a concentrationof 10⁻⁵ M. The amount of ³H-mucins in the spent medium was measured.Each bar represents a mean ±S.E.M. from four culture wells. ATP isindicated as (A). An asterisk (*) indicates a value that issignificantly different (p<0.05) from the control value based on theStudent's t-test for unpaired samples.

[0026]FIG. 7 is a bar graph showing the effects of low molecular weightPLL (20-mer) on mucin release in HTSE cells (Panel A) and on LDH releasein HTSE cells (Panel B). An asterisk (*) indicates a value that issignificantly different (p<0.05) from the control value based on theStudent's t-test for unpaired samples.

[0027]FIG. 8 is a bar graph showing the effects of low molecular weightPLA (20-mer) on mucin release in HTSE cells (Panel A) and on LDH releasein HTSE cells (Panel B). An asterisk (*) indicates a value that issignificantly different (p<0.05) from the control value based on theStudent's t-test for unpaired samples.

[0028]FIG. 9 is a bar graph showing the effects of low molecular weightPLL (14-mer) on mucin release in HTSE cells (Panel A) and on LDH releasein HTSE cells (Panel B). An asterisk (*) indicates a value that issignificantly different (p<0.05) from the control value based on theStudent's t-test for unpaired samples.

[0029]FIG. 10 is a bar graph showing the effects of low molecular weightPLA (14-mer) on mucin release in HTSE cells (Panel A) and on LDH releasein HTSE cells (Panel B). An asterisk (*) indicates a value that issignificantly different (p<0.05) from the control value based on theStudent's t-test for unpaired samples.

[0030]FIG. 11 is a bar graph showing the effects of low molecular weightPLL (5-mer) on mucin release in HTSE cells (Panel A) and on LDH releasein HTSE cells (Panel B).

[0031]FIG. 12 is a bar graph showing the effects of low molecular weightPLA (5-mer) on mucin release in HTSE cells (Panel A) and on LDH releasein HTSE cells (Panel B).

[0032]FIG. 13, Panels A-E are images of rat lung tissue sections showingthe effects of PLA on SO₂ induced metaplasia in rat. Panels A-E showlung tissue from animals that were treated as follows: (A) controluntreated animals; (B) animals treated with SO₂; (C) animals treatedwith both SO₂ and 50 μM PLA (MW 10,800); (D) animals treated with bothSO₂ and 500 μM PLA (MW 10,800); (E) animals treated with both SO₂ and5,000 μM of PLA (MW 10,800).

[0033] Without further elaboration, it is believed that one skilled inthe art can use the preceding description to utilize the presentinvention to its fullest extent. Therefore, the following preferredspecific embodiments illustrate but do not limit the remainder of thedisclosure in any respect.

EXAMPLES

[0034] In the following examples, all parts and percentages are byweight unless otherwise indicated.

Example 1

[0035] Effects of PLA and PLL on mucin release from airway goblet cells.

[0036] The effects of poly-L-arginine (PLA) and poly-L-lysine (PLL) onmucin release from cultured airway goblet cells were evaluated. Tracheasobtained from 7-8 week old male Golden Syrian hamsters (Harlan SpragueDawley, Indianapolis, Ind.) were used to establish a hamster trachealsurface epithelial, (HTSE) cell culture. HTSE cells were harvested andcultured on a thick collagen gel as described in Kim et al., 1989, Exp.Lung Res. 15: 299-314. Mucins were metabolically radiolabeled byincubating confluent cultures (24 well plate, 5×10⁵ cells/well) with 0.2ml/well of a “complete” medium containing 10 μCi/ml of ³H-glucosaminefor 24 hours as described in Kim et al., 1989, Am. J. Resp. Cell Mol.Biol. 1: 137-143. The “complete” medium was prepared by supplementing amixture of Medium 199/Dulbecco's modified Eagle's medium (DME) (1:1)with insulin (5 μg/ml), transferrin (5 μg/ml), epidermal growth factor(12.5 ng/ml), 0.1 μM hydrocortisone, 0.01 μM sodium selenite, 0.1 μMretinoic acid, and 5% fetal bovine serum (Hyclone, Logan, Utah). At theend of the 24 hour incubation period, the spent media (the pretreatmentsample) were collected, and the labeled cultures were washed twice withDulbecco's PBS without Ca⁺⁺ and Mg⁺⁺ and then chased for 30 min in thepresence of varying concentrations of poly-L-arginine (PLA, average MW8,900) or poly-L-lysine (PLL, average MW 9,600). The chased media arereferred to as the treatment samples. PLA and PLL were prepared inphosphate buffered saline (PBS), and the final pH's of these solutionswere adjusted to be between 7.0 and 7.4. PBS solutions within this pHrange do not affect mucin release from HTSE cells; Kim et al., 1989, Am.J. Resp. Cell Mol. Biol. 1: 137-143. At the end of the chase period,floating cells and cell debris were removed from the treatment samplesby centrifigation at 12,000×g for 5 min. Fifty μl of the treatmentsamples were used for lactate dehydrogenase (LDH) assay (as discussed inExample 2 below) and the remaining samples were stored at −80° C. untilassayed for their ³H-mucin contents. High molecular weightglycoconjugates that were excluded after Sepharose CL-4B (Phainacia,Upsaala, Sweden) gel-filtration column chromatography and that wereresistant to hyaluronidase, were defined as mucins, as described in Kimet al., 1985, J. Biol. Chem. 260:4021:4027. Mucins were measured bycolumn chromatography as described in Kim et al, 1987, Proc. Natl. Acad.Sci. USA 84:9304-9308.

[0037] As shown in FIG. 1, both PLA and PLL caused a dose-dependentdecrease in the amount of mucins present in the spent media of the HTSEcell cultures, indicating the inhibition of mucin release by thesepolycationic peptides. Treatment of HTSE cells with varyingconcentrations of PLA resulted in a dose-dependent decrease in theamount of released mucins, reaching an 85% “inhibition” at 10⁻⁵ M (FIG.1). PLL showed a similar pattern of effect with an 87% “inhibition” at10⁻⁵ M (FIG. 1). The decrease in mucin production was not attributableto degradation of mucins during the treatment period or to interferencewith the mucin assay (gel-filtration column chromatography) by thepolycationic peptides. These possibilities were examined using purified³H-mucins that had been prepared as described in Kim et al., 1985, J.Biol. Chem. 260:4021:4027. The pH of the medium was not a contributingfactor to the decrease in mucin production since PLA and PLL wereprepared as 10⁻⁴ M solutions in PBS, with a pH of 7.4. Mucin releasefrom HTSE cells is not affected by this pH (Kim et al., 1989, Am. J.Resp. Cell Mol. Biol. 1: 137-143).

Example 2

[0038] Effects of PLA and PLL on LDH release, ⁵¹Cr release, and cellexfoliation.

[0039] The effects of PLA or PLL on cell toxicity were also examined.The possible cytotoxicity of these polypeptides was assessed by fourdifferent methods: (a) LDH release, (b) ⁵¹Cr release, (c) cellexfoliation, and (d) light microscopy. Cultures were treated with eitherPLA or PLL for 30 min, and then assayed as follows. An LDH assay wasconducted using an LDH assay kit (LD-L10) (Sigma Chemical Co., St.Louis, Mo.), according to the manufacturer's directions. A 51Cr releaseassay was carried out as described in Kim et al., 1987, Proc. Natl.Acad. Sci. USA 84:9304-9308. The degree of exfoliation was measured bycounting the floating cells at the end of the 24 hr post-treatmentperiod. Floating cells were collected as a pellet from spent media bycentrifugation at 200×g for 5 min at 4° C. The resulting cell pellet wassuspended in a solution containing 0.05% Trypsin and 0.02% EDTA, and thesuspension was incubated at 37° C. for 10 min before dissociated cellswere counted using a hemacytometer.

[0040] There was no significant difference between the control and thetreated groups in any of the cytotoxicity assessments. As shown in FIG.2, treatment of HTSE cells with either 10⁻⁵ M PLA or 10⁻⁵ M PLL for 30min caused no significant increases in LDH release. Additionally, thesame concentration of PLA or PLL caused no significant change in ⁵¹Crrelease from HTSE cells: 100±4% for control, 102±11% for PLA, and103±10% for PLL. The number of floating cells/well during the 24 hrpost-treatment period was also not significantly different among thecontrol and treated groups: 1992±132 for control, 2154±84 for cellstreated with 10⁻⁵ M PLA, and 1920±70 for cells treated with 10⁻⁵ M PLL.There was no apparent microscopic difference, using light microscopy,between control and treated HTSE cells, either at the end of the 30 mintreatment period or at the end of the 24 hr post-treatment period.Therefore, in primary HTSE cells, both PLA and PLL do not appear to betoxic at the concentrations that showed an inhibitory effect on mucinproduction.

Example 3

[0041] Determination of whether the inhibitory effects of polycationicpeptides on mucin production is due to the positive charges on thepeptides.

[0042] An examination of whether the inhibitory effects of polycationicpeptides on mucin production is due to the positive charges on thepeptides was conducted using both pharmacological and chemicalapproaches. In one experiment, low molecular weight heparin (LMWH), ahighly negatively charged polysacharride, was added to neutralize thepolycationic peptides. In a second experiment, the polycationic peptideswere N-acetylated to block the positive charges.

[0043] In the first experiment, low molecular weight heparin (LMWH),derived from porcine intestinal mucosa and having an average MW of6,000, was prepared as a solution in PBS, with a final pH of between 7.0and 7.4. The LMWH was added to the cell cultures 5 min prior to theaddition of the polyeationic peptides. As shown in FIG. 3, theinhibitory effect by either 10⁻⁵ M PLA or 10⁻⁵ M PLL on mucin releasewas completely blocked by pretreatment of HTSE cells with 2×10⁻⁵ M LMWH.

[0044] In the second experiment, N-acetylation of the polycationpeptides was performed through a peracetylation reaction. Both PLA andPLL were dissolved in water to a concentration of 11⁻⁴ M and then thesolutions were neutralized to a pH of 7.0 with sodium acetate. Five μlof acetic anhydride was added to 100 μl of this solution, followed byimmediate vortexing, and the resulting solution was incubated for 10 minat room temperature. After repeating the procedures five times, thereaction mixtures were heated at 100° C. for 2 min and were then cooledon ice. For purification of N-acetyl polycationic peptides, 200 μl ofthe reaction mixtures were applied to CM Sepharose CL-6B cation-exchangecolumns (Pharmacia, Upsaala, Sweden, 0.7×5 cm) pre-equilibrated with0.05 M phosphate buffer with a pH of 7.2. The polycationic peptides werefirst eluted from the column with the phosphate buffer, and then with a2 M NaCl solution. Fractions of 320 Al were collected. The absorbance ofeach fraction was measured at 210 nm using a UV spectrophotometer. Asshown in FIG. 4, unacetylated PLA eluted from the column after theaddition of salt (Fractions 13-17) (Panel A), whereas acetylated PLA wascompletely eluted from the column before the salt solution was applied(Fractions 3-8) (Panel B). The same chromatographic pattern was obtainedwith PLL. The size of the peak for acetylated PLA (Fractions 3-8) wasvirtually identical to that of the peak for the original (unacetylated)PLA (Fractions 13-17), indicating the complete N-acetylation of PLA.Peak fractions for both the acetylated and unacetylated forms of thepolycationic polypeptides were separately pooled and then dialyzedagainst PBS. The resultant polypeptides were tested for their effect onmucin release from HTSE cells.

[0045] The “inhibitory” effect of 10⁻⁵ M of PLA or 10⁻⁵ M PLL wascompletely abolished following N-acetylation of these polycationicpeptides (FIG. 5). There was no significant difference between theeffect of the PLA or PLL that had not been column purified or the effectof column-purified PLA or PLL, indicating that the inhibitory effect ofPLA and PLL was not due to contaminants in the preparation. The purityof the original preparation was also apparent based on the profile ofthe cation-exchange column chromatography (FIG. 4).

Example 4

[0046] Inhibition of ATP-induced mucin release by PLA or PLL.

[0047] The effect of polycationic peptides on “stimulated” or“regulated” mucin release was also examined using ATP, which haspreviously been identified as a potent mucin-secretagogue for airwaygoblet cell mucins; Kim et al., 1991, Br. J. Pharmacol., 103:1053-1056.The ATP was prepared as a solution in PBS, with a final pH of between7.0 and 7.4. ATP was added to the cell culture medium, both with andwithout polycationic peptides, during the 30 min chase period.

[0048] The stimulatory effect of ATP was completely blocked in thepresence of either 10⁻⁵ M PLA or 10⁻⁵ M PLL, as shown in FIG. 6. Thepolycationic peptides not only blocked the ATP induced production ofmucins, but also inhibited mucin production in the ATP treated cells tolevels that were almost comparable to levels seen in non-stimulatedcells treated with polycationic peptides. Therefore, a “super”inhibition of mucin was achieved in the ATP treated cells.

Example 5

[0049] Effects of High Molecular Weight PLL or PLA on Mucin Secretion.

[0050] High molecular weight PLL (average MW 78,000;˜533.5 residues) orPLA (average MW 92,000;˜528 residues) were also evaluated for activityin inhibiting mucin secretion from HTSE cells and for toxicity, usingtechniques described in examples 1-4 above. High molecular weight PLLand PLA inhibited mucin secretion in a dose-dependent fashion. However,the use of high molecular weight PLL and PLA was associated withcytotoxicity, as determined by LDH release assays.

Example 6

[0051] Effects of Low Molecular Weight PLL or PLA on Mucin Secretion.

[0052] Low molecular weight PLL or PLA were also evaluated for activityin inhibiting mucin secretion from HTSE cells and for toxicity, usingtechniques described in examples 1-4 above. Low molecular weight PLL (20residues) inhibited mucin secretion in a dose dependent fashion, withoutany evidence of cytotoxicity (FIG. 7). Low molecular weight PLA (20residues) inhibited mucin secretion in a dose dependent fashion, withoutany evidence of cytotoxicity (FIG. 8). Low molecular weight PLL (14residues) inhibited mucin secretion in a dose dependent fashion, withoutany evidence of cytotoxicity (FIG. 9). Low molecular weight PLA (14residues) inhibited mucin secretion in a dose dependent fashion, withoutany evidence of cytotoxicity (FIG. 10). Low molecular weight PLL (5residues) and PLA (5 residues) did not show any significant inhibitionof mucin secretion (FIGS. 11 and 12).

Example 7

[0053] Effects of Inhaled PLL or PLA on SO₂-Induced Goblet CellMetaplasia in Rats.

[0054] To examine the effects of PLL or PLA in vivo, the effect of PLLor PLA on goblet cell metaplasia in rats was determined. Exposure ofrats to SO₂ was carried out essentially according to the methoddescribed in Kase et al, 1982, Arzneim-Forsch Drug Research 32:368-373.Eight to ten week-old male Sprague-Dawley rats were placed in apolyacrylic chamber (100 cm×60 cm×25 cm) and exposed to SO₂ for threeweeks, three hours per day, five days per week. The SO₂ gas wasgenerated from a solution containing 10% (W/V) sodium metabisulfite byaerosolization using an ultrasonic humidifier (Samsung Electronic Co.,Korea). Concentrations of the generated SO₂ gas were monitored beforeand after the exposure using a SO₂ detector kit (Gastec Co., Japan),which has a detection range from 20 to 3600 ppm of SO₂. Theconcentration of the SO₂ gas inside the chamber was maintained at 150ppm during all of the exposure periods.

[0055] PLA (average MW 10,800) was prepared in phosphate buffered saline(PBS), pH 7.2, in various concentrations. Following treatment of theanimals with SO₂ for 2 weeks, animals were treated with both S and PLAduring a third week. A 100 μl aliquot of one of the PLA solutions wasadministered to each rat using an inhaler (PAPI master, Stamberg,Germany), once a day for five consecutive days, during the periodimmediately following the two week SO₂ treatment period. PLA wasadministered between the hours of 10 am to 11 am and SO₂ wasadministered during the hours of 1 pm to 4 pm. At the end of third week,the animals were sacrificed in a CO₂ chamber and the airway tissues wereprocessed for the conventional PAS staining. The histologicalexaminations of the tissue sections (5 μm thick) revealed that gobletcell metaplasia (an increase in the number of purplish epithelial cells)induced by SO₂ could be prevented by PLA in a dose-dependent fashion(FIG. 13).

[0056] The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

[0057] Without further elaboration, it is believed that one skilled inthe art can, using the preceding description, utilize the presentinvention to its fullest extent. The preceding preferred specificembodiments are, therefore, to be construed as merely illustrative, andnot limitative of the remainder of the disclosure.

[0058] The entire disclosure of all patent applications, patents, andpublications cited herein are hereby incorporated by reference.

[0059] From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention and, withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

What is claimed is:
 1. A method of reducing mucin production in theairways of an animal comprising administering at least one polycationicmolecule to the airways of the animal.
 2. A method according to claim 1, wherein at least one polycationic molecule is a polycationic peptide.3. A method according to claim 2 , wherein at least one polycationicpeptide is poly-L-arginine, poly-L-lysine, or a heteropolymer of lysineand arginine.
 4. A method according to claim 2 , wherein the animal istreated for mucin hypersecretion.
 5. A method according to claim 2 ,wherein the animal is treated for asthma, chronic bronchitis, cysticfibrosis, bronchiectasis, or chronic obstructive pulmonary disease.
 6. Amethod according to claim 2 , wherein polycationic peptide isadministered by inhalation.
 7. A method according to claim 3 , whereinthe polycationic peptide is a peptide having 5 to 60 amino acids.
 8. Amethod according to claim 2 , wherein the polycationic peptide is apeptide having 5 to 60 amino acids, wherein at least 50% of the aminoacids are lysine or arginine.
 9. A method according to claim 8 , whereinat least 70% of the amino acids are lysine or arginine.
 10. A methodaccording to claim 9 , wherein at least 90% of the amino acids arelysine or arginine.
 11. A composition comprising at least onepolycationic polypeptide and at least one inhalation adjuvant.
 12. Acomposition according to claim 11 , wherein the inhalation adjuvant is apropellant that is a compressed gas propellant or a liquefied gaspropellant.
 13. A composition according to claim 12 , wherein at leastone polycationic peptide is poly-L-arginine, poly-L-lysine, or aheteropolymer of lysine and arginine.
 14. A composition according toclaim 12 , wherein the polycationic peptide is a peptide having 5 to 60amino acids, wherein at least 50% of the amino acids are lysine orarginine.
 15. A composition according to claim 14 , wherein at least 70%of the amino acids are lysine or arginine.
 16. A composition accordingto claim 15 , wherein at least 90% of the amino acids are lysine orarginine.
 17. A device for administering a polycationic peptide to theairways of an animal comprising an inhalator containing a polycationicpeptide.
 18. A device for administering a polycationic peptide to theairways of an animal according to claim 17 , wherein the polycationicpeptide is contained within a pressurized pack.
 19. A device foradministering a polycationic peptide to the airways of an animalcomprising a pressurized pack containing a composition according toclaim 14 .
 20. A device for administering a polycationic peptide to theairways of an animal comprising a pressurized pack containing acomposition according to claim 15 .